1

BACTERIOLOGICAL ASSESSMENT OF SPOILT TOMATOES

BY

IFEDIEGWU LILIAN ONYINYE

2008483550

A RESEARCH PROJECT WORK SUBMITTED TO THE DEPARTMENT OF

APPLIED MICROBIOLOGY AND BREWING, FACULTY OF
BIOSCIENCES, NNAMDI AZIKIWE UNIVERSITY AWKA IN PARTIAL
FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF BACHELOR
OF SCIENCES (Bsc) DEGREE IN APPLIED MICROBIOLOGY AND
BREWING

SEPTEMBER, 2014
 2

CERTIFICATION

This is to certify that this project was carried out by Ifediegwu Lilian Onyinye

with the Registration Number 2008483550 of the department of Applied

Microbiology and Brewing, Faculty of Biosciences, Nnamdi Azikiwe University,

Awka, under the supervision of Dr. E.J. Archibong for the award of degree of

Bachelor of Science (B.Sc.) in Applied Microbiology and Brewing.

__________________ ____________

DR. E.J. ARCHIBONG DATE

(Supervisor)

____________________ _______________

DR. M. U. ORJI DATE

(Head of Department)

_________________________ _________________

EXTERNAL EXAMINER DATE

 3

DEDICATION

Dedicated to my husband late Mr Egwwonwu Samuel Chijioke.

 4

ACKNOWLEDGEMENTS

I give thanks to Almighty God for seeing me through’.

My gratitude goes to my supervisor Dr, E. J. Archibong for his contribution

towards the success of the project and other staffs of Microbiology and Brewing

Nnamdi Azikiwe University Awka.

My appreciation goes to my dearly parents, Chief and Mrs Gabriel Ifediegwu, and

my beloved husband Late Mr Egwuonwu Samuel Chijioke I know you will be

happy for me wherever you are and also my little Angel Chijioke Samuel victor

Chiziteremu and to all my friends that encouraged me during my difficulties

.Thank you all.

 5

TABLE OF CONTENTS

Title page------------------------------------------------------------------------ i

Certification-------------------------------------------------------------------- ii

Dedication---------------------------------------------------------------------- iii

Acknowledgement----------------------------------------------------------- iv

Table of content--------------------------------------------------------------- v

Abstract------------------------------------------------------------------------ vi

Introduction-------------------------------------------------------------------- 1

Literature Review-------------------------------------------------------------- 3

Varieties of tomatoes---------------------------------------------------------- 5

Cultivation---------------------------------------------------------------------- 6

Handling and Washing of Tomatoes---------------------------------------- 7

Method of the Culturing and Washing of Tomatoes---------------------- 8

Materials and methods--------------------------------------------------------- 9

Samples----------------------------------------------------------------------- 10

Methods----------------------------------------------------------------------- 11

Characterization and identification of isolates-------------------------- 12

Results ---------------------------------------------------------------------- 18

Discussion ------------------------------------------------------------------- 27

Conclusion ------------------------------------------------------------------ 30

References ------------------------------------------------------------------- 31
 6

Appendix ------------------------------------------------------------------------ 33
 7

ABSTRACT

Five samples of Lycopersicon esculentum (tomato) were purchased from two

markets in Awka metropolis and studied to determine their level of bacterial

contamination and safety for human consumption. All the tomatoes were washed

with sterile water, cultured and subcultured in nutrient agar to obtain pure

cultures. 153 bacteria isolates made up of seven species of bacteria found the five

tomatoes samples. Proteus species 83(54.52%), Pseudomonas species 2(1.31%),

Erwinia species 36(23.53%), Salmonella species 7(4.56%), and Shigella species

6(3.92%) were the bacteria species isolated. Pseudomonas species and Erwinia

species were isolated from all the samples. Pseudomonas specie isolates were

significantly higher than other bacteria isolates (p<.05). Viable bacteria counts

from the samples were highest in first sample from Eke market, 9.3x107 cfu/ml,

followed by that of second sample from Eke mkt 9.0x107 cfu/ml and the least

count from Amenyimkt 1.8x107 cfu/ml. Tomatoes contamination with bacteria

was observed in all the three markets; highest in Eke market and least in

Amenyimarket but statistically not significant (P>0.05). Poor processing by

retailers and handling by sellers with unwashed hands was the attributed source of

vegetable contamination with bacteria. Raw vegetables from the markets were

considered unfit for human consumption and adequate cooking with proper

handling before consumption is suggested.

 8

INTRODUCTION

Tomatoes are vegetables which are consumed in relatively small quantities as a

side-dish or as a relish with staple food (Rice, 2002). In order to keep them from

losing their full turgidity, harvested tomatoes often require low temperature and

moist environment during storage (Pierce, 2005). The moist condition of tomatoes

together with low temperature condition encourages the growth of

microorganisms (Schwab, 2008). These conditions also facilitate direct

contamination by microorganisms through the handlers (buyers and sellers).

Indirect contamination may also occur as a result of poor hygienic environment of

the market. Consequently, harvested tomatoes so soon begin to spoil if not sold

immediately. However, cleaning processes reduce the number of microorganisms

(Schwab, 2008). This study of bacteriological quality of tomatoes was aimed at

estimating the level of bacterial contamination of the tomatoes commonly sold in

different markets of Awka metropolis and to determine the safety of the

vegetables for human consumption. Because tomatoes are seasonal crops, has

made their microbiological study necessary so as to identify their spoilage

organisms. Added to their nutritive value, is their medical importance,

inspectional effect as well as economic importance (Frederick, 2003).

Spoilage of tomatoes is defined as those adverse changes in quality of

tomatoes, which are brought about by action of predominantly biological factors.

These changes may include; change in taste, smell and appearance or texture of
 9

the tomatoes fruits (Hooker, 2007). The importance of tomatoes in food industries

and its nutritional importance cannot be over emphasized, and the need for

microbiological examination of tomatoes is very importance as it contributes to a

large extent to economic development. Many of the spoilage organism were

environmental contaminant which have in one time or the other been involved in

food poisoning (Casemore, 2001). Spoiled tomatoes should be disposed and food

handlers should be enlightened on how to handle food products with care as well

as practice hygiene to keep their environment clean. These consumers as well are

adviced not to consumed spoiled tomatoes and should also wash the good ones

properly with clean water before consumption (Beuchat, 2008).

 10

LITERATURE REVIEW

Tomato is the edible, often red fruit berry of the night shade solanium

lycopersium, commonly known as tomato plant. The word tomato comes from the

Spanish tomate, which in turn comes from the Nahuati tomatotl. Tomatoes are

among the vegetable products cultivated world wide that has the greatest

nutritional value (Thompson, 2008). Tomato was erroneously thought to be

poisonous by Europeans who were suspicious of their bright, shiny fruit. Native

version was small like cherry tomatoes and most likely yellow rather than red.

The tomato is native to Western South America and Central America.

By 500 BC it was already being cultivated in Mexico and probably other areas.

Spanish conquistador Hernan Cortes may have been the first to transfer the small

yellow tomatoes to Europe after he captured the Aztec city of Tenochtihan. The

earliest discussion of the tomato in European literature appeared in the herbal

written in 1544 by Pietro Andrea Mattioli an Italian physician and botanist who

suggested that a new type of egg plant had been brought to Italy that was blood

red and golden color and could be divided into segments and eaten like an egg

plant. However it wasn’t until ten years later that tomatoes were named in print

by Mattioli as Pomi d’oro, or golden apple. After the Spanish colonization of

the Americas, the Spanish distribute the tomatoes throughout their colonies in

Caribbean. They also took it to the Philippians from were it spread to South East
 11

Asia and the entire Asian continent. The Spanish also brought the tomato to

Europe. Tomato was not grown in England until 1590s.one of the earliest

cultivators were John Gerard, a barber-surgeon. He believed it was poisonous

though the plant and fruit have low level of tomatine, but are not generally

dangerous.

There are 7500 tomato varieties grown for various purposes. Tomatoes are also

commonly classified as determinate and indeterminate. The determinate bear a

full crop at all once and top off at a specific height. The determinate are preferred

by commercial growers. Indeterminate develop into vine that never top off and

continue producing until killed by frost. They are preferred by home growers and

local market farmers.

VARIETIES OF TOMATOES

Tomato varieties are roughly divided into several categories, based mostly on

shape and sizes. These categories include;

 12

 “Slicing” or “globe” tomatoes are the usual tomatoes of commerce, used

for a wide variety of processing and fresh eating.

 Beefsteak tomatoes are large tomatoes often used for sandwishes and

similar applications. Their kidney bean shape, thinner skin, and shorter

shelf life makes commercial use impractical.

 Oxheart tomatoes can range in size up to beefsteaks, and are shaped like

large strawberries.

 Plum tomatoes or paste tomatoes are bred with higher solice content for

use in tomato sauce and paste and are usually oblong.

 Pear tomatoes are pear shaped are a based upon the San Marzano types for

a richer gourmet paste.

 Cherry tomatoes are small and round, often sweet tomatoes generally

eaten whole in salads.

 Grape tomatoes, in more recent introduction are smaller and oblong, a

variation of plum tomatoes, and used in salad.

 Campari tomatoes are also sweet and noted for their juiceness, low acidity

and lack of mealiness. They are bigger than cherry tomatoes but smaller

than plum tomatoes.

CULTIVATION

The tomato is now grown world-wide for its edible fruits, with thousands of

cultivars having been selected with varying fruit types and for optimum growth in
 13

different grain conditions. Cultivated tomatoes vary in size from tomberries,

about 5mm in diameter, through cherry tomatoes about the same 1 – 2cm sizes as

the white tomatoes, up to beefstake tomatoes 10cm or more in diameter. The most

widely grown commercial tomatoes tend to be in the 5 – 6cm diameter range.

Most cultivars produce red fruits, but a number of cultivars with yellow, orange,

pink, purple, green, black or white fruits are also available. Multi-colored and

stripped fruits can also be quite striking. Tomatoes grown for canning and sauce

are often elongated, 7 – 9cm long and 4 – 5cm diameter. They are known as plum

tomatoes and have a lower water content. Roma type tomatoes are important

cultivars in the sacramento valley.

About 161.8 million tones of tomatoes were produced in the world in 2012. China

the largest producer accounted for about one quarter of the global output followed

by Indian and United States. For one variety, plum or processing tomatoes,

California accounts for 90% of U.S. production and 35% of world production.

HANDLING AND WASHING OF THE TOMATOES

Sterile polythene bags were used to collect and transport the purchased samples to

the Laboratory. A pair of forceps was flamed, allowed to cool, used to pick the

samples and transferred into sterile containers. Sterile distilled water was poured
 14

into these containers. Washing was done by shaking and rocking of the

containers.

METHOD OF CULTURING AND WASHING OF TOMATOES

Serial dilution of the washings was done in which 1ml of each washing was added

into a test-tube containing 9ml of sterile distilled water. Serial tenfold dilution

was carried out from 10-1 to 10-10. This was followed by pour plate technique.
 15

One millilitre aliquot of each dilution from 10-2, 10-5 and 10-7 were cultured into

Nutrient agar, MacConkey agar and Nutrient agar plates respectively. The lid was

replaced in each case. Rocking of these plates were done as soon as the agar were

poured, so as to have the microorganisms evenly separated during growth.

Solidification of the agar was followed by incubation at 370C for 24hrs. After

24hrs, colonies were observed, counted and recorded. Sub culturing of the

Cultures The colonies were purified by sub culturing then in fresh nutrient agar

plates. After purification, the isolates were maintained using nutrient agar slant

and were kept in the refrigerator at 40C for identification.

MATERIALS AND METHOD

The following equipment and materials were used during the course of the

project.

EQUIPMENTS AND MATERIALS

 16

 Autoclave

 Microscope

SAMPLE

5 samples of spoiled fresh tomatoes fruits was obtained from Awka markets,

namely, Eke-awka and Amenyi markets was used for the analysis/ examination.

The sample were collected in sterile polythene bag and transported to the

laboratory immediately for analysis.

MEDIA USED

 Nutrient Agar

 Saburiod dextrose Agar

REAGENTS AND CHEMICALS USED

 Normal saline

 Distilled water

 Acetone

 Lugol’s iodine

 Hydrogen peroxide

 Oxidase reagent (TMPPDH)

 Glucose solution

 Phenol red

 Tryptophan

 Kovac’s reagent
 17

 Potassium hydroxide

 Methylene blue dye

METHODS

PREPARATION OF SAMPLE SOLUTION

The samples bought from different market in Awka; Ekeawka and Amenyi

market) were washed in sterile water to reduce the microbial load, after which a
 18

sterile forceps was used to pull apart the tomatoes, at the site of spoilage, the

monocarp from the advancing margin of the scar were each aseptically transferred

into 10mls of sterile normal saline in various beaker according to the number of

sample collected. The tomatoes were allowed to stand in the normal saline for 10

minutes, before being removed with the sterile forceps from the beakers.

SERIAL DILUTION

Six text tubes containing 9mls of sterile normal saline were placed on a rack on

the bench, using test tube rack. 1ml from the sample solution was pipetted

aseptically into the first test tube and mixed. It was then transferred to the second

test tube and mixed and repeated up to the last tube (10-6). Then 1ml from the last

test tube was discarded.

CULTURING

Sterile Bijou bottles were brought and the respective media, (Saburoid Dextrose

Agar for fungi and nutrient for bacteria growth and Isolation) and prepare. 15mls

of the media were measured into the bijou bottle. It was then autoclaved at 1210C

for 15 minutes. It was then allowed to cool to 450C so as not to kill the organisms

1ml of each dilution were ascetically transferred to a sterile Petri dish. The agar

was then poured and gently rotated. It was then allowed to cool and gel. The

plates were transferred to the incubator and incubated at 370C for 48 hours.

MICROBIAL COUNTING
 19

Microbial count was carried out to determine the microbial concentration in a

given sample from each market and to also compare the amount of growth of

micro-organisms under various conditions (Onyeagba, 2004).

This is done by counting the growth colonies based on their physiological

characteristics and their number on each plate (culture plate) taking note of the

serial diluted tube number. Counts were made from plates that contain between

30-300 colonies.

PREPARATION OF PURE-CULTURE

For pure culture, distinct colonies were aseptically transferred into newly

prepared sterile saburiod dextrose Agar for fungi and Nutrient Agar for bacteria

by streaking, using sterile wire loop, which was later transferred into sterile Agar

slants in bijou bottles, the culture was preserved. Samples for identification tests

were taken from the agar slants. The bacteria culture was kept on nutrient and

slants while fungi cultures were stored on saburiod dextrose agar. The slants were

kept in refrigerator and maintained at 40C.

CHARACTERIZATION AND IDENTIFICATION OF ISOLATES

IDENTIFICATION OF BACTERIAL ISOLATES

 20

Bacterial Isolates were characterized and identified by staining method,

macroscopic examination and biochemical tests. These tests, includes, gram

stains, indole test, catalase test, motility test, citrate utilization test, oxidase test,

starch hydrolysis test and sugar fermentation tests (Bradshaw, 1979).

GRAM STAINING

Gram staining is of great important in the recognition and identification of

bacteria. Grams staining reaction has the widest application, distinguishing nearly

all bacteria as gram positive or gram negative, according to whether or not they

resist decolorization of crystal violet and subsequent treat with iodine. Under this

form of characterization, a thin smear of the bacterial isolates was made on a

glass slide, air, dried and heart fixed by passing it over Bunsen flame for at least

four seconds. The smear was covered with crystal violet stain for 30-60 seconds,

washed of rapidly with clean water. The slides were tipped off all the water and

the smear covered with lugol’s iodine for 30-60 seconds. The iodine washed off

with clean water, decolorized rapidly (few seconds) with acetone and washed of

immediately with clean water. The smear was then covered with safranin for 1

minutes washed off with clean water, the back of the slides were wiped and

placed in a draining rack to be air-dried. The dried smear was examined with

immersion oil microscopically using the (x100) objective lens microscope. Gram-
 21

positive cells appeared purple while the gram negative cells appeared red. (Chees

brough, 2006).

CATALASE TEST

This test is used to differentiate those bacteria that produce the enzyme catalase.

This enzyme acts as a catalyst in the breakdown of hydrogen peroxide to oxygen

and water. In this test, a loopful of each bacterium isolate was aseptically

transferred to clean sterile non vented Petri dish for about two to three days after

which 3% hydrogen peroxide was introduced into the Petri dish was closed and

tithe it, so that the hydrogen peroxide made contact with bacterial growth.

Evolution of oxygen or effervescence as observed production of gas bubbles

indicates a positive result (Cheesbroug, 2006).

OXIDASE TEST

This test is used in differentiating and identification of Pseudomonas, Neisseria,

Vibrio, Brucella, and Pasteurella species all of which produce the enzyme

Cytochrome Oxidase. At the course of carrying out this test, 1.1 gram of the

oxidase reagent tetra methyl-p-phenylenediaminedehydrochoride (Tmppeh) was

dissolved in 10ml of distilled water which was added into a filter paper in a Petri

dish. The inoculum of test culture was smeared into the filter paper using a glass
 22

rod, a positive test was observed of purple colouration occurs within 10 seconds.

(Onyeagba, 2004).

MOTILITY TEST

The hanging drop method was used for this test. A loopful of the bacteria isolates

was introduced into a clean cover slip held between two fingers. A circle was

made with Vaseline on a glass slide. The slide was then placed on top of the cover

slip very well without touching the isolate. The slide was then inverted and

viewed under high power (x40) and oil immersion (x100) objective microscope

(Onyeagba, 2004).

SUGAR UTILIZATION TEST

The sugar solutions used in this experiment were glucose, fructose, lactose and

manitol. The media and each of the sterilization sugar solution were dispersed

into different set of test tubes containing invested Durham tubes. The test

organism was aseptically introduced into the sugar and medium mixed. Also

0.1% phenol red indicator was added into test tubes. (Cheesbrough, 2006).The

indicator and invested Durham tubes were used to detect respectively the

production of acid and liberation of gas by the isolates. Inoculated tubes were

used as control.
 23

INDOLE TEST

Testing for indole production is important in the identification of organism that

are capable of breaking down the amino acid and tryptophan with the release of

indole. The isolated organisms were inoculated into peptone water and incubated

for 48 hours at 370C 0.5ml of kovac’s reagent (p-mettingamionbenzaldehyde)

and gently shaken.Appearance of a reddish color indicated the presence of indole

while no red surface layer indicated absence (Cheesbrough, 2006).

CITRATE UTILIZATION TEST

The test is used in the identification of entrobacteria. This test is based on the

ability of an organism to use citrate as the only source of carbon.At the course of

carrying out this test, a loopful of the culture isolate was streak inoculated into

plates of prepared simmon’s citrate agar. It was incubated at 370C for 72 hours to

observe for citrate as a carbon source was indicated by a change of the agar from

green to deep color blue whereas the negative citrate organism showed neither

growth nor color charge (Onyeagba, 2004).

STARCH HYDROLYSIS

Soluble starch was separately prepared and added to nutrient agar to a 1% soluble

starch medium. The medium was sterilized at 1210C and mg/ cm2 pressure for 15
 24

minutes. It was poured into Petri dishes and allowed to cool. The isolates were

aseptically inoculated on the starch agar plates and inoculated at 370C for 48

hours. After incubation the plates were flooded with iodine and zone of clearance

or hollow around the test organism were observed (Onyeagba, 2004).

VOGES PROSKAUER (V-P) TEST

One milligram of 40% potassium hydroxide and three milligram of 5% alcoholic

naphthol was added to the test tube of glucose and shaken well to observed for

color change within 2-5 minutes. Isolates that produce enough acid during

glucose fermentation were v-p negative (Cheesbrough, 2006).

 25

RESULTS

MACROSCOPIC EXAMINATION

Macroscopic examination of individual spoiled tomatoes showed that some of the

tomatoes have the following features.

 The sample has lost its fitness leading to the loss of its usually shape.

 It has broken epicarp where its juice and seeds gushes out when

compressed.

 Some has black spots at its epicarp.

 It has slightly unpleasant smell.

The total number of bacteria isolates was (5) five in number. The bacteria isolates

from the spoiled samples include; Salmonella species, Proteus species, Erwinia

species, Shigella species and Pseudomonas species (Table 1). All the bacteria

isolates were gram negative, the result of their shown also is their individual

biochemical reaction were given below (Table 4) The bacteria dissimulation the

Isolates were also given in (Table 3).

 26

TABLE 1: AVERAGE CELL COUNTS OF BACTERIAL ISOLATES FROM

THE SPOILED TOMATOES.

__________________________________________________________

S/N Organism Amenyi mkt. Ekeawka mkt.

___________________________________________________________

1Salmonella species 5.3×105Cfu 6.7×105Cfu

2Proteus species 4.7×105Cfu 5.1×105Cfu

3Erwinia species 2.7×105Cfu 9.3×105Cfu

4Shigellaspeciesn 3.1×105Cfu 2.7×105Cfu

5Pseudomonas species 2.9×105Cfu 9.5×105Cfu

______________________________________________________________
 27

TABLE 2: AVERAGE CELL COUNTS OF FUNGI ISOLATES FROM THE

SPOILED TOMATOES OF VARIOUS MARKETS.

___________________________________________________________

S/N Organism Amenyi mkt. Ekeawka mkt.

_____________________________________________________________

1 Cladosporium species 6.0×105Cfu 1.6×105Cfu

2 Aspergillus species 1.4×105Cfu 2.1×105Cfu

3Geotrictium species 1.2×105Cfu 3.4×105Cfu

4 Mucor species 2.9×105Cfu 3.8×105Cfu

5Rhizopus species 3.4×105Cfu 5.4×105Cfu

6Penicillium species 1.5×105Cfu 4.1×105Cfu

_________________________________________________________________

__
 28

TABLE 3: COLONY AND MORPHOLOGICAL CHARACTERISTICS OF

BACTERIAL ISOLATES

Characteristics Organism suspected

Pale coloured colonies with black dots Salmunella spp
scattered rod
Raised surface, round and creamy with Proteus spp
waxy margin rod in chains
Milky mucoid spreading colonies short Erwania spp
rods
Pale coloured colonies that turns to Shigella spp
pink colour when incubated for a long
time, slender rods in chains
Species creamy large irregular edge Pseudomonas spp
colonies in rods chains
 29

TABLE 4: DESCRIPTION OF BIOCHEMICAL PROPERTIES OF BACTERIA

ISOLATED FROM SPIOLED TOMATOES.

S/N Suspected organism 1 2 3 4 5 6 7 8 9 10 11

1 Salmonella species - N + - - - ND - + n -

2Proteus species - + + + + - - - AG - -

3Erwinia species + + + - - ND - + AG + +

4shigella species N + - - - - - - A - -

5 pseudomonas species - + + - + + + - ND A -

_________________________________________________________________

KEYS
 30

+ = positive reaction

- = Negative reaction

A = Acid production

AG = Acid and gas production

ND = Not done

N = Neutral in reaction

G = grams reaction

1= Indole test

2= catalase test

3= Motility test

4= Citrate utilization

5= Oxidize test

6= vogues proskauer

7= Starch hydrolysis

8= Lactose utilization

9= Glucose utilization

10= Fructose utilization

11= Mannitol utilization

 31

IDENTIFICATION OF VARIOUS ISOLATES OBTAINED IN THE

CULTURES

The following biochemical tests were carried out for the characterization and

identification of the organisms, viz: Gram’s stain, Catalase test, Indole test,

Oxidase test, Sugar fermentation test, Motility test, Coagulase test, Citrate

Utilization test, Hydrogen sulphide production test, Voges-Proskauer test, Methyl

red test and haemolytic reaction on blood agar.

ESTIMATION OF INTENSITY OF CONTAMINATION

Intensity of contamination was estimated by using the viable count technique.

Only plates that showed not less than 30 colonies and not more than 300 colonies

were used for this estimation. Colony forming unit (CFU) was used as the

standard unit for the estimation. The bacteria isolates from the tomatoes sample

were made. Salmonella Spp had the highest incidence followed by Proteus Sp

with Shigella, Pseudomonas sp and Erwinia sp recorded the lowest incidence of

each, while Shigella had an incidence.

The bacteria isolates from the samples from Amenyi market are show, 48 bacteria

isolates were made. Salmonella sp had the highest incidence of followed by

Proteus spp 10(22.00%), Erwinia sp 4(7.20%), Pseudomonas sp and Shigella sp

3(6.55%) respectively and Proteus sp 1(1.80%).

A comparison of the bacteria isolates from the two markets of Awka

metropolis. From total of 153 bacteria isolates made from the three markets,
 32

53(34.64%) were from Eke market, 52(33.99%) were from market and

48(31.37%) were Amenyi market. Although the isolates from Eke were slightly

higher than that of Amenyi market, they were not significantly higher (P>0.05)

than that of the Amenyi market.

Of the five bacteria species namely Pseudomonas sp, Shigella sp, Erwinia sp,

Proteus sp, and Salmonella spp. From the three markets, Proteus sp isolates

83(54.25%) were significantly higher (P<0.05) than other bacteria isolates. In the

order of their significance, Proteus sp was followed by Erwinia sp 836(23.53%)

and Pseudomonas sp 15(9.80%).

The distribution of individual bacteria isolates on the sample is shown in table .

Of the five bacteria species, Pseudomonas species and Shigella species were

isolated from all the five samples studied. The highest number of Proteus species

isolates 31(37.75%) were from the first samples from Eke Awka market while the

least 3(3.61%) were from Amenyi. Also the highest number of Erwinia species

isolates 14(38.8%) were from first samples from Eke Awka market samples and

Amenyi market 1(2.78%). Proteus species was not isolated from Amenyi market

second sample, it has the highest isolate 2(50.0%)Erwinia species and

Pseudomonas species were also not isolated from Amenyi market and Eke

market. The highest number of Erwinia species. isolates 8(42.86%) were from

Eke market and the least 3(20.0%) . Also the highest member of Pseudomonas

specie isolate 3(50.0%) were from Eke market and the least 1(16.67%).
 33

Salmonella specie was not isolated from Amenyi market only. The highest

number of Proteus specie isolates 3(50.0%) were from Eke market and least

number 1(14.29%). With the exception of Pseudomonas species with its highest

number of isolates tomatoes, five bacteria species namely Erwinia specie,

Pseudomonas specie, Proteus specie. Shigella specie and Salmonella specie had

their highest isolates from Eke market samples. An equal number of isolates of

Erwinia species was isolated from Eke market and Amenyi market (one isolate

from each). All the five bacteria isolates were made from Eke market and Amenyi

market while only Proteus specie and Erwinia specie were found.

The percentage distribution of the isolates on the tomatoes samples is represented

in figure 1. Of the 153 bacteria isolates, 61(39.87%) were from Eke market first

samples 42(27.45%) from Eke market second samples, 38(24.84%) from Amenyi

market first sample, 8(5.23%) from Amenyi market second sample and

4(2.61%) . The distribution of the isolates on the different samples was

statistically significant (P<0.05).

The viable bacteria counts from the different tomato samples bought from

markets of Awka metropolis were

DISCUSSION

In Awka Markets, the commonly sold vegetables such as Lycopersicum

esculentum were found to be contaminated with bacteria. This contamination

could be as a result of poor processing methods which may involve washing with
 34

fecal contaminated stream water and not washing at all. Though washing with

water gives fresh appearance to the vegetables and delays decomposition but it

could add microorganisms, especially, psychrotrophs from water or ice (Frazier,

2005). The washing provides moist surface that encourages bacteria growth on

longer storage. Also tomatoes are subject to contamination by containers like the

baskets unless adequately sanitized (Bessie, 2007) .

The bacteria isolates from the vegetable samples were Proteus species,

Pseudomonas species, Erwinia specie, and Salmonella specie. The distribution of

these isolates varied with different vegetables. Proteus species was found to have

highcount while low count was recorded too. Erwinia species ranked next to

Pseudomonas specie in high count and it was recorded also. Shigella species was

the isolate with the least count. The low bacteria count may be due to anti-

microbial compounds in them which are inhibitory to multiplication of

microorganisms (Tindall, 2002). Distribution of bacteria isolates also differed in

the different markets. Eke market recorded the highest bacteria count in Proteus

species while Amenyi market recorded the least count in this isolate. No isolate of

Pseudomonas specie was found in Amenyi market samples and Eke markets

yielded very low count for Pseudomonas species.

 35

The higher incidences of Salmonella species over others may be due to its spores

which could resist killing by high temperature of ultra-violet sun rays which may

kill and reduce the bacteria load in vegetables during exposure and display for

sale. High incidence of Salmonella specie may probably be due to handling by

buyers and sellers whose hands may have been contaminated with faecal matters

or they may have been contaminated from the farm yards when fertilized with

human and animal manures. Bacillus species has been implicated as one of the

common contaminants of vegetables (Banwart, 2001). The presence of

Pseudomonas specie and Shigella specie could probably be due to washing of

these vegetables with water contaminated with faecal matter. Low incidences of

Lactobacillus acidophilus may be attributed to their inability to produce spores

like Bacillus species. Being acidophilic, their survival and multiplication require

acidic and relatively anaerobic conditions, which are not provided for on the

tomatoes.

All the bacteria isolated were opportunistic pathogens, in that; they usually cause

infection if suitable opportunity arises. This suitable opportunity could be seen in

a person with weak natural immunity probably due to poor health, malnutrition,

infection with HIV or drug therapy.

 36

CONCLUSION

Adequate care should be taken in processing tomatoes to destroy the

microorganisms before they enter inside the human body and obtain favourable
 37

conditions which could support their increase in number; thereby causing food-

borne disease or food poisoning to the detriment of the human health.

REFERENCES

Banwart, G.J. (2001). Bacteria as food spoilage organisms. Basic food

microbiology. Avi Publishing Co. Westport. Conn. Pp. 119-125.

 38

Bessie. E., Breadley, H. and Sundberg, C. (2007). Keeping food safe. Double

day and Company I Gardk.

Beuchat, L.R (2008). Surface contamination of fruits and vegetables eaten raw.

Journals of Food Safety 98:2-10.

Bradshaw, F.B (1979). Diagnosis and management of food borne illness. A

Primer or Physicians. 2:1-69.

Casemore, O.P (2001).food borne protozoa infection. 4th edition. Edward Arnold

Press, London. Pp 100-119.

Cheesbrough, M.L (2006). Fruit and vegetables processing of tomatoes. 3 rd

edition, University Collage Press, London. Pp 301-310.

Fredrick, J.O (2007). Small scale fruit and vegetable processing and products. 4th

edition. Elsevier Applied Science Press, London, pp23-58.

Frazier, W.C. and Westhoff. D.C. (2005). Food microbiology. McGraw-Hill

Publishing Company Ltd., New Delhi pp. 217-240.

Hooker, A.G (2007). Consumers of processed fruit and vegetables products.

Journals of Indian Food Industry, 16(3):25-36.

Onyeagba, K.K (2004). Fruits and vegetable spoiled by microorganism. Journals

of Queensland. 21(8):560-568.

Peirce, L.C. (2005). Vegetables: Characteristics, production and marketing.

John Wiley, New York. Pp. 55-90.

 39

Rice, R.P., Rice L.W., and Tyndall, H.D (2002). Fruit and Vegetable

production in Africa. (2nd edition). Macmillan Press Ltd., Hong Kong, Pp.

8-12.

Schwab, A.H., Duran, A.P., Barnard, R.J., and Read, R.B. (2008). Microbial

quality of some spices and herbs in Retail markets. Journal Applied and

Environmental Microbiology 24 (3): 627-630.

Tindall, H.D. (2002). Vegetablesinthetropics. Liverpool University Press, Hong

Kong. P. 21.

Thompson, T.L (2008). Maintaining mango fruit quality during the export chain.

Food Reservation International. 44:1254-1263.

APPENDIX

MEDIA
 40

Nutrient agar:

A clean filter paper was placed on the weighing balance and the weight of the

paper is measured, after which a clean spatula was used to collect the dehydrated

media onto the clean paper on the weighing balance until the media weighed 7

grams for nutrient agar was measured out. The container of the dehydrated media

was closed immediately after collection. The weighed out media was dissolved in

250 mms of distilled water from clean glass. The dissolution was in water by

gently rotation in the conical flask. After complete dissolution of the media the

flask was covered with cotton wool and foil before autoclave for 15 minutes at

1210c and 15 psi. After autoclaving the sterilized media where poured into Petri

dishes in a sterile environment, allowed to gel. The gelled media were preserved

in the refrigerator. The plates is incubated for 35 to 37.0c and kept for senility

check.

SABUROID DEXTROSE AGAR

A clean fitter just as above but 15.5 grams was measured out and dissolved in

250ml, The dissolution was in water by gently rotation in the cornical flask. After

complete dissolution of the media the flask was covered with cotton wool and foil

before autoclave for 15 minutes at 1210c and 15 psi. After autoclaving the

sterilized media where poured into Petri dishes in a sterile environment, allowed
 41

to gel. The gelled media were preserved in the refrigerator. The plates is

incubated for 35 to 37.0c and kept for senility check.